In patients with multiple sclerosis (MS), there is a distinct gender bias, with approximately twice as many affected females as males. Sex hormones may contribute to susceptibility or resistance to MS by influencing development and function of potentially pathogenic T cells specific for central nervous system (CNS) antigens, as well as regulatory T cells that might modify the course of disease. Previously, we reported that low doses of 17beta-estradiol (E2) can reduce severity of EAE by inhibiting activation, cytokine and chemokine production, and encephalitogenicity of murine T cells specific for myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), or myelin basic protein (MBP), and by inhibiting I recruitment of inflammatory cells into the CNS. Of particular importance, estrogen treatment profoundly reduced intracellular production of TNF-alpha, known as a critical inflammatory cytokine in EAE and MS. Recent evaluation of E2 effects on EAE using microarray analysis of splenocyte gene expression confirmed potent inhibition of TNF-alpha, and further identified several previously unsuspected immune-associated candidate genes appearing to be strongly affected by E2 treatment in vivo. These modulatory effects of estrogen on pathogenic, recruited, and regulatory cells in EAE are likely mediated y direct interaction with estrogen receptors (ER), which include the classical ER-alpha and ER-beta that are internal f receptors (iER), and possibly membrane ERs (mER) that may be distinct from iERs. This proposal will test the hypothesis that modulation of EAE by estrogen involves receptor-mediated regulation of TNF-alpha and several other novel immunerelated genes, thereby inhibiting inflammatory effects of macrophages, dendritic cells and T cells and enhancing regulatory NKT cell activity. Our primary goals are to determine if the inhibitory effects of E2 on EAE can be observed in spontaneous EAE in the absence of regulatory T and NKT cells, and if inhibitory effects of E2 on EAE are mediated through iER-alpha and /or iERbeta, or in contrast, by neither of the classical iERs. Using newly developed ER-alpha and ER-beta knockout mice we will for the first time associate E2- dependent regulation with either or both iERs, or alternatively with mER if E2 effects persist in double KO mice. Moreover, for each novel E2-affected gene implicated from the microarray analysis, we will investigate the cellular source, effects of E2 on transcription and protein production, and contribution to EAE induction in vivo. Genes found to be important in EAE will be further evaluated in blood cells from women with different levels of E2. The work proposed will identify key estrogen-sensitive genes, including TNFalpha, contributing to EAE induction and resistance. Changes in these genes can be followed during estrogen treatment as surrogate markers to verify effective doses of estrogen. From this research, we will develop a solid basis for using estrogen replacement therapy for MS.